Platelet Factor 4 (PF4) is a small molecular weight protein that is secreted from the .alpha.-granules of platelets when the cells undergo the release reaction. This phenomenon occurs when the platelets become activated subsequent to contact with subendothelial tissue and a variety of other phsyiological agents including thrombin, ADP and epinephrine. Platelet factor 4 is quantitated using classical radioimmunoassay techniques, Thrombosis Research 8, pg 51-58, (1976) and Thrombosis Research 11, pg 673-686, (1977).
Platelet factor 4 (PF4) whether radiolabeled or not, will bind to specific antiserum. In a mixture of labeled and unlabeled (.sup.125 I-PF4 and PF4) protein, each will be bound to a limited amount of antiserum in proportion to its concentration in the mixture. When PF4 from plasma, serum or a standard solution is equilibrated with .sup.125 I-PF4 and PF4 antiserum, the amount of .sup.125 I-PF4 that is bound to the antiserum will be inversely proportional to the amount of nonradioactive PF4 present in the sample or standard solution. By separating the antiserum complex from unbound PF4 and measuring the radioactivity of .sup.125 I-PF4 bound in the complex, concentrations of added PF4 can be plotted versus % of .sup.125 I-PF4 bound. A standard curve can then be used to determine the amount of PF4 in the patient samples.
Stable standard samples are essential for reliable quantitation of platelet factor 4. Unexpectedly, it has been found that heparin, Merck Index, 9th Ed., 4510, stabilizes platelet factor 4 standard samples and labeled reagent. Heparin is a highly sulfated dextrorotatory mucopolysaccharide having specific anticoagulant properties. It is composed of D-glucosamine and D-glucuronic acid residues and has a molecular weight of about 20,000. Heparinic acid, physiologically acceptable alkaline metal and alkaline earth cationic salts such as sodium, potassium, lithium, magnesium, calcium or ammonium and the like, as well as other sulfated polysaccharides such as sulfated pectins and dextrins having heparin like activity are suitable sources for heparin activity for practicing the present invention.
Platelet factor 4 is a platelet specific protein with heparin neutralizing activity which is liberated during the platelet release reaction as measured by heparin thrombin clotting time test, Thrombosis Research 8, pg. 51-58, (1976).